Pinctada martensii anti-low temperature trait related snp molecular marker and application thereof

Abstract

The application provides a SNP molecular marker related to low-temperature resistance of Pinctada martensii and application thereof, and belongs to the technical field of aquatic genetic breeding. The molecular marker comprises one or more of SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, SNP456, SNP718, SNP823 and SNP831. The molecular marker can be used for genotype identification of parent shellfish before seed breeding, and the low-temperature resistance of offspring population can be improved by screening parent shellfish with multiple advantageous genotypes.

Description

Technical Field

[0001] This invention belongs to the field of aquatic genetic breeding technology, and in particular relates to a SNP molecular marker related to the low-temperature resistance trait of Pinctada martensii and its application. Background Technology

[0002] Marine mollusks are poikilothermic animals, lacking the ability to regulate their body temperature, which is the same as the temperature of their environment. Therefore, ambient temperature is one of the most important environmental factors affecting the life activities of mollusks, directly influencing their survival, growth, metabolism, reproduction, and development, and determining their geographical distribution. The Pinctada fucata martensii, also known as the Hepu pearl oyster, belongs to the class Bivalvia of the phylum Mollusca. In my country, it is mainly distributed in the South China Sea, including Guangdong, Guangxi, and Hainan. It is a typical warm-water mollusk with a suitable temperature range of 15–30℃, and an optimal growth temperature of 23–28℃. It has a weak tolerance to low temperatures; a drop in water temperature of 6–8℃ for more than 21 hours will result in mass mortality. Therefore, cultivating low-temperature tolerant strains of the Pinctada fucata is a prerequisite for expanding pearl oyster farming to the north and broadening the pearl oyster farming area.

[0003] Traditional breeding methods suffer from drawbacks such as long breeding cycles, high investment, and high time and labor costs. Screening for DNA molecular markers related to low-temperature resistance for molecular-assisted breeding is crucial for accelerating the genetic improvement of cold resistance. A review of existing domestic and international literature on this technology has yielded no research reports on the association between the Perlucin and α-Tubulin genes and the low-temperature resistance of Pinctada martensii, nor any reports on the correlation between polymorphic sites of these two genes and the low-temperature resistance of Pinctada martensii. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide an SNP molecular marker related to the low-temperature resistance trait of Pinctada martensii and its application. This series of molecular markers can accurately identify Pinctada martensii with low-temperature resistance.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a SNP molecular marker related to the low-temperature resistance trait of Pinctada martensii, wherein the molecular marker includes one or more of SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, SNP456, SNP718, SNP823, and SNP831.

[0007] Wherein: SNP351 is located at position 351 of the Perlucin gene, and the polymorphism is C or T;

[0008] The SNP352 is located at position 352 of the Perlucin gene and has a polymorphism of G or T.

[0009] The SNP362 is located at position 362 of the Perlucin gene and has a polymorphism of A or G.

[0010] The SNP365 is located at position 365 of the Perlucin gene and has a polymorphism of C or T.

[0011] The SNP367 is located at position 367 of the Perlucin gene and has a polymorphism of A or G.

[0012] The SNP378 is located at position 378 of the Perlucin gene and has a polymorphism of A or T.

[0013] The SNP396 is located at position 396 of the Perlucin gene and has a polymorphism of A or G.

[0014] The SNP456 is located at position 456 of the Perlucin gene and has a polymorphism of A or G.

[0015] The SNP718 is located at position 718 of the α-Tubulin gene and has a polymorphism of G or T.

[0016] The SNP823 is located at position 823 of the α-Tubulin gene, and its polymorphism is either A or C.

[0017] The SNP831 is located at position 831 of the α-Tubulin gene and has a polymorphism of A or G.

[0018] Preferably, the nucleotide sequence of the Perlucin gene is shown in SEQ ID NO.1.

[0019] Preferably, the nucleotide sequence of the α-Tubulin gene is shown in SEQ ID NO.2.

[0020] This invention provides a primer set for amplifying the aforementioned SNP molecular markers, the primer set comprising one or more of the following sites:

[0021] Table 1 SNP molecular markers and their primer sets

[0022]

[0023]

[0024] The present invention provides a kit comprising one or more of the primers described above.

[0025] This invention provides the application of the SNP molecular marker, the primer set, or the kit in the identification of low-temperature resistance in Pinctada martensii, for the purpose of identifying the genotype of low-temperature resistance loci in Pinctada martensii.

[0026] This invention provides a method for evaluating the low-temperature resistance of Pinctada martensii, comprising the following steps:

[0027] (1) Extract genomic DNA from the sample to be tested;

[0028] (2) Using the genomic DNA from step (1) as a template, perform PCR amplification using the primer set;

[0029] (4) Perform agarose gel electrophoresis or molecular sequencing on the amplification products to obtain the genotype of the molecular marker;

[0030] (5) Based on the genotyping results, determine the low-temperature resistance trait of the sample to be tested.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention is the first to discover the Perlucin gene and α - The Tubulin gene is associated with low-temperature tolerance in Pinctada martensii. For the first time, it was found that SNP351 CC, SNP352 TT, SNP362 GG, SNP365 CC, SNP367 AG, SNP378 TT, SNP396 AA, and SNP456 AG in the Perlucin gene and SNP718 GT, SNP823 AC, and SNP831 AG in the α-Tubulin gene are dominant genotypes for low-temperature tolerance in Pinctada martensii.

[0033] The primer sets designed according to one or more of SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, SNP456, SNP718, SNP823, and SNP831 can identify the genotype of parent shellfish before seedling breeding. By screening parent shellfish with multiple advantageous genotypes, the cold tolerance of offspring populations can be improved. Attached Figure Description

[0034] Figure 1 Schematic diagram of electrophoresis results for SNP351 site.

[0035] Figure 25 shows the sequencing peaks of the samples. Detailed Implementation

[0036]

[0037] This invention provides primer sets for the SNP molecular markers, including one or more of the following in Table 2:

[0038] Table 2 SNP molecular markers and their primer sets

[0039]

[0040]

[0041] In this invention, the primer sets Perlucin-F and Perlucin-R are designed using Perlucin containing the molecular markers SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, and SNP456; and the α-Tubulin-F and α-Tubulin-R are designed using α-Tubulin containing the molecular markers SNP718, SNP823, and SNP831.

[0042] This invention provides a kit comprising one or more of the primers described above. The kit also includes DNA polymerase, 10× polymerase chain reaction (PCR) buffer, a mixture of deoxyribonucleoside triphosphate (dNTPs), and magnesium chloride. This kit is used to identify the low-temperature resistance trait of *Pinctada martensii*.

[0043] This invention provides the application of the SNP molecular markers or primer sets in the identification of low-temperature resistance traits in Pinctada martensii, for the identification of genotypes at low-temperature resistance loci. This invention can identify the dominant genotypes of low-temperature resistant breeding lines in Pinctada martensii as follows: In the Perlucin gene, the dominant genotypes are SNP351 CC, SNP352 TT, SNP362 GG, SNP365 CC, SNP367 AG, SNP378 TT, SNP396 AA, and SNP456 AG; in the α-Tubulin gene, the dominant genotypes are SNP718 GT, SNP823 AC, and SNP831 AG. If the Pinctada martensii sample tested exhibits one or more of the above dominant genotypes, the sample is considered a candidate parent for low-temperature resistant breeding lines, and individuals with a higher proportion of these dominant genotypes are ultimately selected as breeding parents.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1: Screening of SNP molecular markers related to low-temperature resistance in Pinctada martensii

[0046] 1. Obtaining the low-temperature resistant strain:

[0047] 1200 healthy Pinctada martensii oysters were selected from the basic population (constructed from the wild population W in Beibu Gulf) in Zhanjiang and transferred to Nan'ao Island in Shantou for cultivation (minimum water temperature 10-11℃). The individuals that survived the winter were used as parents. The parents were artificially bred to cultivate a low-temperature resistant selective line F1. Following the same route to obtain F1, low-temperature resistant selective lines F2 and F3 were cultivated. Seedling cultivation and adult cultivation were carried out in a conventional manner to form the low-temperature resistant selective line RF3.

[0048] 2. Genomic DNA extraction:

[0049] (1) 30 individuals were randomly selected from RF3 and the wild population W in Beibu Gulf. They were anesthetized with magnesium carbonate, and a small amount of gill tissue was cut out, placed in cryovials, flash-frozen with liquid nitrogen, and stored in a -80℃ freezer for later use.

[0050] (2) Genomic DNA was extracted from the gill tissue of each individual in step (1) according to the instructions of the Marine Animal Tissue Genomic DNA Extraction Kit (TIANGEN). The specific operation steps are as follows:

[0051] 1) Cut no more than 30 mg of adductor muscle tissue and place it in a centrifuge tube containing 200 μL of GA buffer. Vortex for 15 seconds.

[0052] 2) Add 4 μL of RNase A (100 mg / mL) solution, shake for 15 seconds, and let stand at room temperature for 5 minutes.

[0053] 3) Add 20 μL of Proteinase K (20 mg / mL) solution, vortex to mix, and briefly centrifuge to remove water droplets from the inner wall of the cap. Incubate at 56°C until the tissue is completely dissolved, briefly centrifuge to remove water droplets from the inner wall of the cap, and then proceed to the next step.

[0054] 4) Add 200 μL of buffer GB, invert thoroughly to mix, incubate at 70°C for 10 min, the solution should become clear, and then briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0055] 5) Add 200 μl of anhydrous ethanol and mix thoroughly by inverting. At this point, flocculent precipitate may appear. Briefly centrifuge to remove water droplets from the inner wall of the tube cap.

[0056] 6) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0057] 7) Add 500 μL of buffer GD (add anhydrous ethanol before use) to the adsorption column CB3, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0058] 8) Add 600 μL of washing buffer PW (add anhydrous ethanol before use) to the adsorption column, centrifuge at 12,000 rpm for 30 seconds, discard the waste liquid, and put the adsorption column CB3 back into the collection tube.

[0059] 9) Repeat step 8).

[0060] 10) Place the adsorption column CB3 back into the collection tube, centrifuge at 12000 rpm for 2 min, centrifuge at 12000 rpm for 30 s, discard the waste liquid, and place the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material.

[0061] 11) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μL of elution buffer TE to the middle of the adsorption membrane, incubate at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 30 seconds, and collect the solution into the centrifuge tube to obtain the individual's genomic DNA.

[0062] 3. Whole genome resequencing

[0063] The quality of the DNA samples extracted in step 2 was assessed using a NanoDrop-1000 ultra-micro UV spectrophotometer and gel electrophoresis. The integrity of the extracted DNA samples was assessed using 1% agarose gel electrophoresis. The NanoDrop-1000 was used to determine the DNA concentration and the levels of protein and RNA contamination within the DNA. After passing genomic DNA testing, the samples from 60 individuals in the RF3 and W populations were sent to BGI Genomics in Wuhan for sequencing using an Illumina HiSeq™ 2000 platform. Whole-genome resequencing databases for RF3 and W were obtained through sequencing.

[0064] 4. Determine the locations of the Perlucin and α-Tubulin genes in the whole genome of Pinctada martensii.

[0065] Based on the Perlucin and α-Tubulin gene sequences of Pinctada martensii shown in SEQ ID NO:1 and SEQ ID NO:2, the above gene sequences were compared with the fine genome map of Pinctada martensii using BioEdit software to obtain the corresponding positions of the Perlucin and α-Tubulin gene sequences in the whole genome of Pinctada martensii.

[0066] 5. Extract relevant molecular marker sites

[0067] In the whole-genome resequencing database of each sample from the RF3 and W populations, the bases at positions 351, 352, 362, 365, 367, 378, 396, and 456 of the Perlucin gene sequence and positions 718, 823, and 831 of the α-Tubulin gene sequence were extracted using UltraEdit from the corresponding genomic positions obtained in step 4 above.

[0068] 6. Manually count the genotypes of the above 11 SNP sites in the Perlucin and α-Tubulin genes in each sample.

[0069] 7. The association between the low-temperature tolerance of Pinctada martensii and the genotypes of 11 SNP loci was analyzed using the chi-square test in SPSS 22.0. The results are shown in Table 3.

[0070] Table 3. Association analysis results between low-temperature tolerance and 11 SNP genotypes.

[0071]

[0072] The association analysis results in Table 3 show that the genotype frequencies of Perlucin gene SNP351 (CC), SNP352 (TT), SNP362 (GG), SNP365 (CC), SNP367 (AG), SNP378 (TT), SNP396 (AA), and SNP456 (AG) and α-Tubulin gene SNP718 (GT), SNP823 (AC), and SNP831 (AG) were significantly higher in RF3 than in the W population. This indicates that these genotypes are significantly associated with the low-temperature tolerance trait of Pinctada martensii and can be used as markers for auxiliary selection of the low-temperature tolerance trait in Pinctada martensii.

[0073] Example 2: Design of SNP molecular marker primers and determination of SNP locus genotype

[0074] 1. Design of SNP molecular marker primers

[0075] Based on the Perlucin gene sequences containing gene loci SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, and SNP456, and the α-Tubulin gene sequences containing gene loci SNP718, SNP823, and SNP831, specific amplification primers were designed for different loci using Primer 5.0 software and manual selection. The primer sequences for each locus are shown in Table 2.

[0076] 2. Determination of SNP locus genotype: Genotype analysis of the loci was performed using PCR technology with a four-primer amplification blocked mutation system.

[0077] (1) Extraction of genomic DNA: Extract genomic DNA from the sample according to step 2 of Example 1.

[0078] (2) PCR amplification: The DNA of the sample was amplified using the designed primer sequences. The amplification system consisted of 15 μL of the following: 0.2 μL DNA polymerase (5 U / μL), 1.5 μL 10× polymerase chain reaction (PCR) buffer, 1.2 μL deoxyribonucleoside triphosphate (dNTP) mixture (2.5 mM each), 0.4 μL magnesium chloride (25 mM), 0.3 μL each of the forward and reverse outer primers, 1.5 μL each of the forward and reverse inner primers, and 6.6 μL double-distilled water. The program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension 1:10 min, for a total of 35 cycles, 72℃ extension for 10 min, and storage at 4℃ to obtain the amplified product.

[0079] (3) Detection of amplification products and determination of SNP loci genotype: The amplification products were detected by 1% agarose gel electrophoresis. The electrophoretic bands in the gel pattern were observed to determine the genotype of the sample.

[0080] Taking SNP351 as an example, SNP351-F1 and SNP351-R2 are positive controls, with an amplified fragment of 357 bp; SNP351-F2 and SNP351-R2 have amplified fragments of 153 bp; and SNP351-F1 and SNP351-R1 have amplified fragments of 252 bp. The amplified products were detected using 1% agarose gel electrophoresis. In the gel pattern, only a 357 bp band indicates the absence of a mutation site. Bands of 357 bp and 252 bp indicate TT homozygotes, bands of 357 bp and 153 bp indicate CC homozygotes, and bands of 357 bp, 252 bp, and 153 bp indicate CT heterozygotes. See details... Figure 1 A schematic diagram of the electrophoresis results for SNP351 is shown. Then, based on the analysis results in Table 3, the dominant genotypes at each locus are identified to determine whether the sample is a low-temperature resistant breeding line. The determination of other loci follows the same logic.

[0081] Example 3: Design of SNP molecular marker primers and determination of SNP locus genotype

[0082] 1. Design of SNP molecular marker primers

[0083] Based on the Perlucin gene sequences containing gene loci SNP351, SNP352, SNP362, SNP365, SNP367, SNP378, SNP396, and SNP456, and the α-Tubulin gene sequences containing gene loci SNP718, SNP823, and SNP831, specific amplification primers were designed using Primer 5.0 software and manual selection. The primer sequences are shown in Table 4.

[0084] Table 4 Amplification Primers

[0085]

[0086] 2. Determination of SNP locus genotype: Sequence analysis of the amplification products.

[0087] (1) Extraction of genomic DNA: Extract genomic DNA from the sample according to step (2) of Example 1.

[0088] (2) PCR amplification: The DNA of the sample was amplified using the designed primer sequences. The amplification system consisted of 10 μL of 10 μL of ddH2O, 0.4 μL of genomic DNA, 0.4 μL of upstream specific primer F, 0.4 μL of downstream specific primer R, and 5 μL of LimeSTAR. The program was set as follows: 95℃ pre-denaturation for 5 min; 98℃ denaturation for 15 s, 59℃ annealing for 30 s, 72℃ extension for 3 min, for a total of 35 cycles, followed by a 10 min extension at 72℃, and storage at 4℃ to obtain the amplified product.

[0089] (3) Genotype of SNP site: The amplification product was sent to Guangzhou Sangon Biotech Co., Ltd. for sequencing to obtain the amplified sequence information.

[0090] Sequencing results showed that the product obtained by the amplification reaction using specific primers Perlucin-F and Perlucin-R was the Perlucin gene sequence containing the above 8 SNP sites, as shown in SEQ ID NO:1. The genotype of the sample was determined by detecting bases at positions 351, 352, 362, 365, 367, 378, 390, 396, and 456 bp of this sequence. The product obtained by the amplification reaction using specific primers α-Tubulin-F and α-Tubulin-R was the α-Tubulin gene sequence containing the above 3 SNP sites, as shown in SEQ ID NO:2. The genotype of the sample was determined by detecting bases at positions 718, 823, and 831 bp of this sequence.

[0091] Based on the above technical solution, five individual samples of Pinctada martensii from the F3 generation of the low-temperature resistant breeding population were identified. The identification results are shown in Table 5 and [Table 6]. Figure 2 From Table 5 and Figure 2The results showed that all five sequenced individuals possessed six superior genotypes of the Perlucin gene: CC at SNP351, TT at SNP352, GG at SNP362, CC at SNP365, TT at SNP378, and AA at SNP396. This indicates that these five individuals are associated with the low-temperature tolerance trait of Pinctada martensii and can be used as parents to breed offspring according to conventional Pinctada martensii seedling techniques.

[0092] Table 5. Genotyping results of sequencing at 8 SNP sites in the Perlucin gene.

[0093]

[0094] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A SNP molecular marker associated with low-temperature resistance in Pinctada martensii, characterized in that, The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.1; the 351st base of the SNP molecular marker is C or T, the 352nd base is G or T, the 362nd base is A or G, the 365th base is C or T, the 378th base is A or T, and the 396th base is A or G.

2. A primer set for amplifying the SNP molecular marker as described in claim 1, characterized in that, The primer set is shown below: 。 3. A reagent kit, characterized in that, Includes the primer set as described in claim 2.

4. The application of the primer set as described in claim 2 or the kit as described in claim 3 in the identification of low-temperature resistance in Pinctada martensii, characterized in that, The genotypes of low-temperature resistance loci in Pinctada martensii are used to identify the following genotypes: CC at position 351, TT at position 352, GG at position 362, CC at position 365, TT at position 378, and AA at position 396. In these cases, Pinctada martensii is identified as having low-temperature resistance.

5. A method for evaluating the low-temperature resistance of Pinctada martensii, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Using the genomic DNA from step (1) as a template, perform PCR amplification using the primer set as described in claim 2; (4) Perform agarose gel electrophoresis or molecular sequencing on the amplification products to obtain the genotype of the molecular marker; (5) Based on the genotyping results, determine the low-temperature resistance trait of the sample to be tested. If the genotype at position 351 of SEQ ID NO.1 is CC, the genotype at position 352 is TT, the genotype at position 362 is GG, the genotype at position 365 is CC, the genotype at position 378 is TT and the genotype at position 396 is AA, then the Pinctada martensii is identified as having the low-temperature resistance trait.

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